Intranasal administration of the growth-compromised HSV-2 vector DeltaRR prevents kainate-induced seizures and neuronal loss in rats and mice

Abstract

Identification of targets and delivery platforms for gene therapy of neurodegenerative disorders is a clinical challenge. We describe a novel paradigm in which the neuroprotective gene is the herpes simplex virus type 2 (HSV-2) antiapoptotic gene ICP10PK and the vector is the growth-compromised HSV-2 mutant DeltaRR. DeltaRR is delivered intranasally. It is not toxic in rats and mice. ICP10PK is expressed in the hippocampus of the DeltaRR-treated animals for at least 42 days in the absence of virus replication and late virus gene expression. Its expression is regulated by an AP-1 amplification loop. Intranasally delivered DeltaRR prevents kainic acid-induced seizures, neuronal loss, and inflammation, in both rats and mice. The data suggest that DeltaRR is a promising therapeutic platform for neurodegenerative diseases.

Fig 1

ΔRR does not replicate nor cause cell death in neurons. (A) Single step growth curves of HSV-1, HSV-2, ΔPK and ΔRR in OHC were done as described in materials and methods. (B) OHC infected with HSV-2 (panel 1) or ΔRR (panels 2 and 3) as in (A) were stained with ethidium homodimer (cell death) at 96 hr p.i. (panels 1 and 2) and counterstained with DAPI (panel 3). (C) Ethidium staining cells in (B) were counted and the results are expressed as % dead cells ± SEM. (D) ΔRR infected OHC were stained with C₁₂FDG and the % staining cells in the dentate gyrus (DG) and the CA1 and CA3 hippocampus fields calculated relative to DAPI staining as described in materials and methods.

Fig. 2

ICP10PK-LacZ reaches the hippocampus through the lateral olfactory bulb tract. (A) Schematic representation of the tract with relevant connections. Asterisk indicates tissues examined for ICP10PK and found to be positive. (B) Immunohistochemical staining of brain tissue along the lateral olfactory bulb tract with ICP10 antibody.

Fig. 3

ICP10PK is expressed in the hippocampus after intranasal ΔRR instillation. (A) C57BL/6 mice were given ΔRR (3 doses; 3×10⁶ pfu each) and extracts of nasal epithelia (lanes 1-3), and hippocampi (lanes 4-7) were immunoblotted with antibody to ICP10, VP5 and actin. Blots were stripped and reprobed between each antibody. (B) Fold increase in ICP10PK-LacZ expression was determined by densitometric scanning. Results are ICP10PK-LacZ/actin ratios (N.E.= nasal epithelia; Hipp = hippocampi). (C) Mice treated with ΔRR or PBS as in (A) were given KA (30mg/kg, i.p.) 24 hours after the last treatment (day 0). Extracts of olfactory bulbs (lanes 1, 2) and hippocampi (lanes 4, 5) collected on day 8 were immunoblotted with ICP10 antibody and the blots were sequentially stripped and re-probed with antibodies to VP5 and actin. Extracts of HSV-2 infected Vero cells (MOI=2; 24 hrs p.i.) (lane 3) served as control. (D) Brains from animals treated with ΔRR as in (A) were harvested on days 2 and 42 after the final treatment, coronally sectioned and stained by immunohistochemistry with ICP10 antibody (panel 1) or preimmune IgG (panel 4) or with FITC-conjugated ICP10 antibody (panels 2, 3). The % staining cells in the latter sections was calculated relative to the total number of cells determined by DAPI in 3 randomly selected fields of 29μm² (at least 250 cells) from 5 serial sections for all animals. Results are % ICP10PK-LacZ + cells ± SEM. Similar results were obtained in Sprague Dawley rats. (E) Brains from animals treated with PBS (panels 1,2) or ΔRR (panels 3,4) as in (A) were harvested on day 42, coronally sectioned and thionin stained.

Fig. 4

Intranasally delivered ΔRR protects from KA induced seizures. Sprague Dawley rats and C57BL/6 mice were given 3 intranasal doses of ΔRR or ΔPK (5×10⁶ pfu and 3×10⁶ pfu each for rats and mice, respectively) or PBS, and given of KA (15mg/kg and 30mg/kg for rats and mice, respectively) 24 hrs later by i.p. injection. They were examined for behavioral changes for 5 hours and rated on a scale of: 0, normal; 1, catatonic staring and immobilization; 2, ‘wet-dog shakes’, abnormal ambulation, stretching of limbs; 3, rearing and falling behavior; 4, tonic-clonic seizure activity; 5, death. Average behavioral score ± SEM is presented for each hour of observation, for rats (A) and mice (B). The % animals in each treatment group experiencing a behavioral score = 4 at any time during the observation period is shown for rats (C) and mice (D).

Fig. 5

ΔRR prevents KA-induced neuronal loss and oxidative stress. Sprague Dawley rats were treated with PBS (panels 1,4) ΔRR (panels 2,5), or ΔPK (panels 3,6) and given KA as in Fig. 2. Coronal sections of brains collected 2 days later were stained with thionin (A). The numbers of neurons were counted in 3 randomly selected fields of 29μm² (at least 250 cells) from 5 serial sections for all animals and the data are expressed as % neuronal loss ± SEM relative to untreated brains (B). Duplicate sections were stained with FITC-conjugated ICP10 antibody (C). To visualize oxidative stress, coronal sections were stained with NITT (D). Similar results were obtained in mice for the CA3 region.

Fig. 6

ΔRR inhibits KA-induced astrogliosis and microglia activation. (A) C57BL/6 mice were treated with ΔRR, ΔPK, or PBS and given KA as in Fig. 2. Hippocampi were collected on days 0, 4, and 8 and extracts were immunoblotted with GFAP antibody, stripped and re-probed with antibody to actin. Fold increase was determined by densitometric scanning and results are presented as GFAP/actin ratio normalized to untreated. (B) Brains from mice treated with ΔRR, ΔPK, or PBS and given KA as in Fig. 2, were harvested on days 4 and 6, sectioned coronally, and serial sections were stained with antibodies to TNFα or CD11b. Day 8 sections were negative.

Fig. 7

ICP10PK expression is regulated by an AP-1 feedback amplification loop. (A) Sprague Dawley rats were treated with ΔRR or PBS and given KA (15mg/kg; i.p.) at 24 hrs after the final treatment (day 0). Extracts of olfactory bulbs (day 8) were immunoblotted with P-c-Jun antibody. Blots were stripped and re-probed with antibody to total c-Jun. (B) Neuronally differentiated PC12 cells were infected with ΔRR (MOI=2) and extracts obtained at 8 hrs p.i. were immunoblotted with P-c-Jun followed by c-Jun antibodies. (C) Neuronally differentiated PC12 cells were transfected with pJW24 or pJZ34 (2 μg) using Fugene 6 transfection reagent and infected (or not) with ΔRR (MOI=2) at 24 hrs post transfection. CAT expression was measured by ELISA 24 hrs later and results are expressed as ng/ml (D). OHC treated (2hrs, 37°C) with ΔRR, UV-inactivated ΔRR (10⁵ pfu), or mock treated with PBS and exposed to KA (5μM, 24 hrs) on day 2 were stained C₁₂FDG (ICP10PK-LacZ expression) and ethidium homodimer (dead cells) and the number of ethidium homodimer staining cells counted as described in Materials and Methods. The % positive cells ± SEM was calculated relative to DAPI staining.

Fig. 8

Schematic representation of the ICP10 gene and ΔRR construction. (A) In ΔRR, the ribonucleotide reductase (RR) domain was replaced with the ß-galactosidase gene (LacZ); in ΔPK, the protein kinase (PK) domain was deleted. Both mutants retain the transmembrane (TM) and extracellular (EC) domains and amino acids 13-26, which are recognized by the ICP10 antibody. (B) The RR sequences in the ICP10 expression vector (pJW17) were replaced with LacZ and flanking HSV-2 DNA sequences from plasmid p101 were added. The ICP10PK-LacZ chimera was introduced into HSV-2 by recombination and ΔRR virus was isolated as blue plaques after staining with X-gal.